Cell surface-engineering to embed targeting ligands or tracking agents on the cell membrane.

The key challenge to improve the efficacy of cell therapy is how to efficiently modify cells with a specific molecule or compound that can guide the cells to the target tissue. To address this, we have developed a cell surface engineering technology to non-invasively modify the cell surface. This technology can embed a wide variety of bioactive molecules on any cell surface and allow for the targeting of a wide range of tissues in a variety of disease states. Using our cell surface engineering technology, mesenchymal stem cells (MSC)s were modified with: 1) a homing peptide or a recombinant protein to facilitate the migration of the cells toward a specific molecular target; or 2) magnetic resonance imaging (MRI) contrast agents to allow for in vivo tracking of the cells. The incorporation of a homing peptide or a targeting ligand on MSCs facilitated the migration of the cells toward their molecular target. MRI contrast agents were successfully embedded on the cell surfaces without adverse effects to the cells and the contrast agent-labeled cells were detectable by MRI. Our technology is a promising method of cell surface engineering that is applicable to a broad range of cell therapies.

Oligopeptide complex for targeted non-viral gene delivery to adipocytes.

Commercial anti-obesity drugs acting in the gastrointestinal tract or the central nervous system have been shown to have limited efficacy and severe side effects. Anti-obesity drug development is thus focusing on targeting adipocytes that store excess fat. Here, we show that an adipocyte-targeting fusion-oligopeptide gene carrier consisting of an adipocyte-targeting sequence and 9-arginine (ATS-9R) selectively transfects mature adipocytes by binding to prohibitin. Injection of ATS-9R into obese mice confirmed specific binding of ATS-9R to fat vasculature, internalization and gene expression in adipocytes. We also constructed a short-hairpin RNA (shRNA) for silencing fatty-acid-binding protein 4 (shFABP4), a key lipid chaperone in fatty-acid uptake and lipid storage in adipocytes. Treatment of obese mice with ATS-9R/shFABP4 led to metabolic recovery and body-weight reduction (>20%). The ATS-9R/shFABP4 oligopeptide complex could prove to be a safe therapeutic approach to regress and treat obesity as well as obesity-induced metabolic syndromes.

Recent advances in microbial and enzymatic engineering for the biodegradation of micro- and nanoplastics.

This review examines the escalating issue of plastic pollution, specifically highlighting the detrimental effects on the environment and human health caused by microplastics and nanoplastics. The extensive use of synthetic polymers such as polyethylene (PE), polyethylene terephthalate (PET), and polystyrene (PS) has raised significant environmental concerns because of their long-lasting and non-degradable characteristics. This review delves into the role of enzymatic and microbial strategies in breaking down these polymers, showcasing recent advancements in the field. The intricacies of enzymatic degradation are thoroughly examined, including the effectiveness of enzymes such as PETase and MHETase, as well as the contribution of microbial pathways in breaking down resilient polymers into more benign substances. The paper also discusses the impact of chemical composition on plastic degradation kinetics and emphasizes the need for an approach to managing the environmental impact of synthetic polymers. The review highlights the significance of comprehending the physical characteristics and long-term impacts of micro- and nanoplastics in different ecosystems. Furthermore, it points out the environmental and health consequences of these contaminants, such as their ability to cause cancer and interfere with the endocrine system. The paper emphasizes the need for advanced analytical methods and effective strategies for enzymatic degradation, as well as continued research and development in this area. This review highlights the crucial role of enzymatic and microbial strategies in addressing plastic pollution and proposes methods to create effective and environmentally friendly solutions.

Adsorption behavior of triclosan on microplastics and their combined acute toxicity to D. magna.

Microplastics (MP) have been recently identified as emerging water contaminants in worldwide. Owing to its physicochemical properties, MP have been considered as a vector of other micropollutants and may affect their fate and ecological toxicity in the water environment. In this study, triclosan (TCS), which is a widely-used bactericide, and three frequently found types of MP (PS-MP, PE-MP, and PP-MP) were investigated. The adsorption behavior of TCS on MP was investigated by the effect of reaction time, initial concentration of TCS, and other water chemistry factors. Elovich model and Temkin model are the most fitted well with kinetics and adsorption isotherms, respectively. The maximum TCS adsorption capacities were calculated for PS-MP (9.36 mg/g), PP-MP (8.23 mg/g), and PE-MP (6.47 mg/g). PS-MP had higher affinity to TCS owing to hydrophobic and π-π interaction. The TCS adsorption on PS-MP was inhibited by decreasing concentrations of cations, and increasing concentration of anion, pH, and NOM concentration. At pH 10, only 0.22 mg/g of adsorption capacity was obtained because of the isoelectric point (3.75) of PS-MP and pKa (7.9) of TCS. And almost no TCS adsorption occurred at NOM concentration of 11.8 mg/L. Only PS-MP had no acute toxic effect on D. magna, whereas TCS showed acute toxicity (EC50,24h of TCS = 0.36 ± 0.4 mg/L). Although survival rate increased when TCS with PS-MP due to lower the TCS concentration in solution via adsorption, PS-MP was observed in intestine and body surface of D. magna. Our findings can contribute to understanding the combined potential effects of MP fragment and TCS to aquatic biota.

Low-molecular-weight methylcellulose-based thermo-reversible gel/pluronic micelle combination system for local and sustained docetaxel delivery.

PURPOSE: To develop low-molecular-weight methylcellulose (LMw MC)-based gel/Pluronic F127 micelle combination system for local and sustained delivery of docetaxel (DTX). METHODS: LMw MC and Pluronic F127 were used to formulate an injectable thermo-reversible gel/micelle combination system containing DTX. The DTX-loaded combination system was characterized and its therapeutic efficacy evaluated in a subcutaneous tumor model. RESULTS: Mixtures of LMw MC, AS, and Pluronic F127 formed gel at ~15-40°C depending on AS concentration. The combination system released DTX for >30 days with a biphasic and sustained release pattern, and DTX stability was maintained during release. The combination system significantly enhanced anti-cancer effects of DTX and prolonged survival of the model mouse in comparison with free DTX. CONCLUSIONS: The LMw MC gel/Pluronic F127 micelle combination system constitutes a promising tool for reducing tumor size and eradicating remaining tumor cells before and after surgery.

The Potential of Topoisomerase Inhibitor-Based Antibody-Drug Conjugates.

DNA topoisomerases are essential enzymes that stabilize DNA supercoiling and resolve entanglements. Topoisomerase inhibitors have been widely used as anti-cancer drugs for the past 20 years. Due to their selectivity as topoisomerase I (TOP1) inhibitors that trap TOP1 cleavage complexes, camptothecin and its derivatives are promising anti-cancer drugs. To increase accumulation of TOP1 inhibitors in cancer cells through the targeting of tumors, TOP1 inhibitor antibody-drug conjugates (TOP1-ADC) have been developed and marketed. Some TOP1-ADCs have shown enhanced therapeutic efficacy compared to prototypical anti-cancer ADCs, such as T-DM1. Here, we review various types of camptothecin-based TOP1 inhibitors and recent developments in TOP1-ADCs. We then propose key points for the design and construction of TOP1-ADCs. Finally, we discuss promising combinatorial strategies, including newly developed approaches to maximizing the therapeutic potential of TOP1-ADCs.

Targeted delivery of heat shock protein 90 inhibitors prevents growth of HER2-positive tumor.

Heat shock protein 90 (HSP90) plays a crucial role in the survival of cancer cells. When an inhibitor blocks the signaling pathway of HSP90, its client proteins are degraded, destabilized, and inactivated. Although HSP90 inhibitors are in various clinical trials, there are no HSP90 inhibitor-immunoconjugates due to the difficulty in chemical modification of HSP90 inhibitors. Here we show that biological affinity binding enables the incorporation of HSP90 inhibitors to an antibody without the need for chemical conjugation. We constructed a recombinant fusion protein composed of an anti-HER2 scFv and an HSP90 inhibitor-binding domain (HER2 scFv-HBD). The HBD spontaneously captures a HSP90 inhibitor, resulting in the formation of an HER2 scFv-HBD/HSP90 inhibitor complex. In an HER2-positive cancer mouse model, targeted delivery of HSP90 inhibitors was confirmed and improved anti-cancer efficacy was observed. We have proven the promise of tumor-directed HSP90 inhibition as a new form of targeted therapy.

Peptide Adjuvant to Invigorate Cytolytic Activity of NK Cells in an Obese Mouse Cancer Model.

Cancer patients who are overweight compared to those with normal body weight have obesity-associated alterations of natural killer (NK) cells, characterized by poor cytotoxicity, slow proliferation, and inadequate anti-cancer activity. Concomitantly, prohibitin overexpressed by cancer cells elevates glucose metabolism, rendering the tumor microenvironment (TME) more tumor-favorable, and leading to malfunction of immune cells present in the TME. These changes cause vicious cycles of tumor growth. Adoptive immunotherapy has emerged as a promising option for cancer patients; however, obesity-related alterations in the TME allow the tumor to bypass immune surveillance and to down-regulate the activity of adoptively transferred NK cells. We hypothesized that inhibiting the prohibitin signaling pathway in an obese model would reduce glucose metabolism of cancer cells, thereby changing the TME to a pro-immune microenvironment and restoring the cytolytic activity of NK cells. Priming tumor cells with an inhibitory the prohibitin-binding peptide (PBP) enhances cytokine secretion and augments the cytolytic activity of adoptively transferred NK cells. NK cells harvested from the PBP-primed tumors exhibit multiple markers associated with the effector function of active NK cells. Our findings suggest that PBP has the potential as an adjuvant to enhance the cytolytic activity of adoptively transferred NK cells in cancer patients with obesity.

Transplantation of mesenchymal stem cells within a poly(lactide-co-epsilon-caprolactone) scaffold improves cardiac function in a rat myocardial infarction model.

AIMS: Cardiac tissue engineering has been proposed as an appropriate method to repair myocardial infarction (MI). Evidence suggests that a cell with scaffold combination was more effective than a cell-only implant. Nevertheless, to date, there has been no research into elastic biodegradable poly(lactide-co-epsilon-caprolactone) (PLCL) scaffolds. The aim of this study was to investigate the effect of mesenchymal stem cells (MSCs) with elastic biodegradable PLCL scaffold transplants in a rat MI model. METHODS AND RESULTS: Ten days after inducing MI through the cryoinjury method, a saline control, MSC, PLCL scaffold, or MSC-seeded PLCL scaffold was transplanted onto the hearts. Four weeks after transplantation, cardiac function and histology were evaluated. Transplanted MSCs survived and differentiated into cardiomyocytes in the injured region. Left ventricular ejection fraction in the MSC+PLCL group increased by 23% compared with that in the saline group; it was also higher in the MSC group. The infarct area in the MSC+PLCL group was decreased by 29% compared with that in the saline group; it was also reduced in the MSC group. CONCLUSION: Mesenchymal stem cells plus PLCL should be an excellent combination for cardiac tissue engineering.

One-Step Method for Instant Generation of Advanced Allogeneic NK Cells.

Conventional combinatorial anticancer therapy has shown promising outcomes; still, a significant interest in developing new methods to reinforce and possibly merge chemotherapy and immunotherapy persists. Here, a new one-step method that immediately modifies immune cells into a targeted form of chemoimmunotherapy through spontaneous and rapid incorporation of hydrophobized antibody-drug conjugates (ADCs) on the surface of immune cells is presented. Therapeutic objectives of this approach include targeted delivery of a potent chemotherapeutic agent to avoid adverse effects, enhancing the mobilization of infused immune cells toward tumor sites, and preserving the intense cytotoxic activities of immune cells against tumor cells. The embedding of hydrophobized ADCs on the immune cell membrane using the strategy in this study provides noninvasive, nontoxic, and homogenous modifications that transiently arm immune cells with highly potent cytotoxic drugs targeted toward cancer cells. The resulting surface-engineered immune cells with ADCs significantly suppress the tumor growth and drive the eradication of target cancer cells through combinatorial anticancer effects. This novel strategy allows convenient and timely preparation of advanced chemoimmunotherapy on a single immune cell to treat various types of cancer.

Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson's disease models.

An excess of reactive oxygen species (ROS) relative to the antioxidant capacity causes oxidative stress, which plays a role in the development of Parkinson's disease (PD). Because mitochondria are both sites of ROS generation and targets of ROS damage, the delivery of antioxidants to mitochondria might prevent or alleviate PD. To transduce the antioxidant protein human metallothionein 1A (hMT1A) into mitochondria, we computationally designed a cell-penetrating artificial mitochondria-targeting peptide (CAMP). The recombinant CAMP-conjugated hMT1A fusion protein (CAMP-hMT1A) successfully localized to the mitochondria. Treating a cell culture model of PD with CAMP-hMT1A restored tyrosine hydroxylase expression and mitochondrial activity and reduced ROS production. Furthermore, injection of CAMP-hMT1A into the brain of a mouse model of PD rescued movement impairment and dopaminergic neuronal degeneration. CAMP-hMT1A delivery into mitochondria might be therapeutic against PD by alleviating mitochondrial damage, and we predict that CAMP could be used to deliver other cargo proteins to the mitochondria.

Direct Incorporation of Functional Peptides into M-DNA through Ligand-to-Metal Charge Transfer.

Conventional nonviral gene delivery methods suffer from the toxicity of the cationic nature of polymeric carriers. There is a significant need for a new method of gene delivery that overcomes the limitations and allows targeted gene delivery. In this study, we have developed a new method to incorporate functional peptides into DNA without the need for chemical conjugations by utilizing a ligand-to-metal charge transfer (LMCT) transition, which occurs between divalent metal ions and the sulfhydryl group in cysteine. To apply the LMCT transition to the incorporation of cysteine-containing targeting peptides into DNA, divalent metal ions must be first introduced to DNA. Zn2+ ions spontaneously intercalate into the DNA base pairs in the pH range of 7.0-8.5, resulting in the conversion of normal B-DNA to metal-bound DNA (M-DNA). We found that the Zn2+ ions present in M-DNA could interact with the sulfhydryl groups in cysteines of targeting peptides through the LMCT transition, and the M-DNA/peptide complex could specifically transfect the target cells.

Development of porous PLGA/PEI1.8k biodegradable microspheres for the delivery of mesenchymal stem cells (MSCs).

Multipotent mesenchymal stem cells (MSCs) promise a therapeutic alternative for many debilitating and incurable diseases. However, one of the major limitations for the therapeutic application of human MSC (hMSC) is the lengthy ex vivo expansion time for preparing a sufficient amount of cells due to the low engraftment rate after transplantation. To solve this conundrum, a porous biodegradable polymeric microsphere was investigated as a potential scaffold for the delivery of MSCs. The modified water/oil/water (W1/O/W2) double emulsion solvent evaporation method was used for the construction of porous microspheres. PEI1.8k was blended with poly(lactic-co-glycolic acid) (PLGA) to enhance electrostatic cellular attachment to the microspheres. The porous PLGA/PEI1.8k (PPP) particles demonstrated an average particle size of 290µm and an average pore size of 14.3µm, providing a micro-carrier for the MSC delivery. PPP particles allowed for better attachment of rMSCs than non-porous PLGA/PEI1.8k (NPP) particles and non-porous (NP) and porous PLGA (PP) microspheres. rMSC successfully grew on the PPP particles for 2weeks in vitro. Next, PPP particles loaded with 3 different amounts of hMSC showed increased in vivo engraftment rates and maintained the stemness characteristics of hMSC compared with hMSCs-alone group in rats 2weeks after intramyocardial administration. These customized PPP particles for MSC delivery are a biodegradable and injectable scaffold that can be used for clinical applications.

Intracellular transduction of TAT-Hsp27 fusion protein enhancing cell survival and regeneration capacity of cardiac stem cells in acute myocardial infarction.

Myocardial infarction (MI) results in the substantial loss of functional cardiomyocytes, which frequently leads to intractable heart disorders. Cardiac stem cells (CSCs) that retain the capacity to replace all cardiac cells might be a promising strategy for providing a source of new functional cardiomyocytes; however, the poor survival and engraftment of transplanted CSCs in the hostile environment of MI critically mitigate their therapeutic benefits. To capitalize their therapeutic potential, an ex vivo strategy in which CSCs were introduced to the recombinant heat shock protein 27 (Hsp27) through a TAT protein transduction domain for increasing the viability and engraftment in the infarcted myocardium was designed. A recombinant TAT fused Hsp27 (TAT-Hsp27) was able to enter CSCs in a dose-dependent manner. CSCs transduced with TAT-Hsp27 expressed not only endogenous Hsp27 but externally introduced Hsp27, resulting in substantial increase of their anti-oxidative and anti-apoptotic properties via suppressing reactive oxygen species production, the MAPKs signaling pathway, and caspase activation. TAT-Hsp27 enabled CSCs to be protected from apoptotic- and hypoxic-induced cell death during in vitro cardiomyogenic differentiation. In vivo studies demonstrated that CSCs transduced TAT-Hsp27 significantly increased the survival and engraftment in the acutely infarcted myocardium, which is closely related to caspase activity suppression. Finally, CSCs transduced TAT-Hsp27 improved cardiac function and attenuated cardiac remodeling in comparison with non-transduced CSCs. Overall, our approach, which is based on the ex vivo intracellular transduction of TAT-Hsp27 into CSCs before myocardial delivery, might be effective in treating MI.

Protective effects of protein transduction domain-metallothionein fusion proteins against hypoxia- and oxidative stress-induced apoptosis in an ischemia/reperfusion rat model.

Ischemic heart diseases caused by insufficient oxygen supply to the cardiac muscle require pharmaceutical agents for the prevention of the progress and recurrence. Metallothionein (MT) has a potential as a protein therapeutic for the treatment of this disease due to its anti-oxidative effects under stressful conditions. In spite of its therapeutic potential, efficient delivery systems need to be developed to overcome limitations such as low transduction efficiency, instability and short half-life in the body. To enhance intra-cellular transduction efficiency, Tat sequence as a protein transduction domain (PTD) was fused with MT in a recombinant method. Anti-apoptotic and anti-oxidative effects of Tat-MT fusion protein were evaluated under hyperglycemia and hypoxia stress conditions in cultured H9c2 cells. Recovery of cardiac functions by anti-apoptotic and anti-fibrotic effects of Tat-MT was confirmed in an ischemia/reperfusion (I/R) rat myocardial infarction model. Tat-MT fusion protein effectively protected H9c2 cells under stressful conditions by reducing intracellular ROS production and inhibiting caspase-3 activation. Tat-MT fusion protein inhibited apoptosis, reduced fibrosis area and enhanced cardiac functions in I/R. Tat-MT fusion protein could be a promising therapeutic for the treatment of ischemic heart diseases.

Sequential delivery of TAT-HSP27 and VEGF using microsphere/hydrogel hybrid systems for therapeutic angiogenesis.

Ischemic disease is associated with high mortality and morbidity rates, and therapeutic angiogenesis via systemic or local delivery of protein drugs is one potential approach to treat the disease. In this study, we hypothesized that combined delivery of TAT-HSP27 (HSP27 fused with transcriptional activator) and VEGF could enhance the therapeutic efficacy in an ischemic mouse model, and that sequential release could be critical in therapeutic angiogenesis. Alginate hydrogels containing TAT-HSP27 as an anti-apoptotic agent were prepared, and porous PLGA microspheres loaded with VEGF as an angiogenic agent were incorporated into the hydrogels to prepare microsphere/hydrogel hybrid delivery systems. Sequential in vitro release of TAT-HSP27 and VEGF was achieved by the hybrid systems. TAT-HSP27 was depleted from alginate gels in 7 days, while VEGF was continually released for 28 days. The release rate of VEGF was attenuated by varying the porous structures of PLGA microspheres. Sequential delivery of TAT-HSP27 and VEGF was critical to protect against muscle degeneration and fibrosis, as well as to promote new blood vessel formation in the ischemic site of a mouse model. This approach to controlling the sequential release behaviors of multiple drugs could be useful in the design of novel drug delivery systems for therapeutic angiogenesis.

Hypoxic resistance of hypodermically transplanted pancreatic islets by using cell-absorbable antioxidant Tat-metallothionein.

Subcutaneous site is ideal for clinical islet transplantation because it has the advantage of simple operation procedure under local anesthesia and can be biopsied when needed. However, the transplantation outcomes at subcutaneous site have been disappointing due to hypoxia-induced oxidative stress by poor vascularization. We hypothesized that subcutaneously transplanted islets would have hypoxia resistance by using internalization of metallothionein (MT), an antioxidant scavenging enzyme, which was mediated by fusion between MT and cell penetrating Tat peptide. The Tat-MT was dose-dependently transduced into islets without any damage. Tat-MT-treated islets could be protected from oxidative stress induced by intracellular nitric oxide donor, sodium nitroprusside (SNP). When Tat-MT-treated islets were subcutaneously transplanted into diabetic nude mice, they normally controlled the blood glucose levels without severe fluctuation (median survival time (MST): >30 days), whereas most untreated islets were rejected (MST 17 days). From the intraperitoneal glucose tolerance test 5 days after posttransplantation, glucose responsiveness of Tat-MT-treated islets was similar to that of normal healthy mice, while untreated islets had delayed glucose responsiveness. From the results of immunohistochemical stain, Tat-MT-treated islets had strong anti-insulin positive cells and lower anti-HIF-1α positive cells. However, untreated islets had rare anti-insulin positive cells and strong anti-HIF-1α-positive cells. Collectively, these findings demonstrated that Tat-MT delivery into islet could offer a new strategy for successful islet transplantation under subcutaneous space.

Injectable microsphere/hydrogel hybrid system containing heat shock protein as therapy in a murine myocardial infarction model.

Heat shock proteins, acting as molecular chaperones, protect heart muscle from ischemic injury and offer a potential approach to therapy. Here we describe preparation of an injectable form of heat shock protein 27, fused with a protein transduction domain (TAT-HSP27) and contained in a hybrid system of poly(d,l-lactic-co-glycolic acid) microsphere and alginate hydrogel. By varying the porous structure of the microspheres, the release of TAT-HSP27 from the hybrid system was sustained for two weeks in vitro. The hybrid system containing TAT-HSP27 was intramyocardially injected into a murine myocardial infarction model, and its therapeutic effect was evaluated in vivo. The sustained delivery of TAT-HSP27 substantially suppressed apoptosis in the infarcted site, and improved the ejection fraction, end-systolic volume and maximum pressure development in the heart. Local and sustained delivery of anti-apoptotic proteins such as HSP27 using a hybrid system may present a promising approach to the treatment of ischemic diseases.

Dual-mode enhancement of metallothionein protein with cell transduction and retention peptide fusion.

Protein transduction domains (PTDs), also known as cell-penetrating peptides (CPPs), have been developed as effective systems for delivering bio-active cargos such as proteins, genes and particles. Further improvements on cell-specific targeting, intracellular organelle targeting and intracellular retention are still necessary to enhance the therapeutic effect of PTD fusion proteins. In order to enhance the cell transduction and retention of anti-oxidative metallothionein protein (MT), MT was recombinantly fused with transcriptional activator (Tat) with or without a short peptide (sMTS) derived from mitochondria malate dehydrogenase (mMDH). Cellular uptake and retention time of fusion protein were significantly increased in the H9c2 cell by sMTS. The Tat-sMTS-MT (TMM) fusion protein protected H9c2 cells more effectively against hypoxia, hyperglycemia and combination compared with Tat-MT (TM) by reducing intracellular ROS level. It maintained the normal blood glucose level over an extended period of time in a streptozotocin-induced diabetic mouse model. PTD-sMTS-MT fusion protein has a potential to be used as a therapeutic protein for the treatment or prevention of diabetes and diabetic complications.

Intracellular organelle-targeted non-viral gene delivery systems.

Gene therapy is a rapidly growing approach for the treatment of various diseases. To achieve successful gene therapy, a gene delivery system is necessary to overcome several barriers in the extracellular and intracellular spaces. Polymers, peptides, liposomes and nanoparticles developed as gene carriers have achieved efficient cellular uptake of genes. Among these carriers, cationic polymers and peptides have been further developed as intracellular organelle-targeted delivery systems. The cytoplasm, nucleus and mitochondria have been considered primary targets for gene delivery using targeting moieties or environment-responsive materials. In this review, we explore recently developed non-viral gene carriers based on reducible systems specialized to target the cytoplasm, nucleus and mitochondria.